Introduction

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Diadenosine polyphosphates (abbreviated to Ap_nA, where n = 2-7) are natural compounds. In particular diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), diadenosine pentaphosphate (Ap₅A), Ap₆A, and Ap₇A are found in exocytotic vesicles, such as those in nerve terminals, adrenal medullary chromaffin cells, and platelets (Flodgaard and Klenow, 1982; Rodriguez del Castillo et al., 1988; Pintor et al., 1992a,b,c, 1997; Schlütter et al., 1994; Pintor and Miras-Portugal, 1995; Jankowski et al., 1999), and diadenosine pyrophosphate (Ap₂A) has recently been identified in secretory granules of cardiac myocytes (Luo et al., 1999). They have diverse actions in peripheral and central tissues because of their roles as extracellular signal molecules (Hoyle, 1990; Pintor et al., 1996; Kisselev et al., 1998; Hoyle et al., 2001).

Ap₄A and Ap₅A have been found in rabbit tears (Pintor et al., 2002b), and both of these dinucleotides together with Ap₃A have been isolated from human tears (Pintor et al., 2002a,b). In rabbits, topical corneal application of Ap₄A or Ap₅A, but not Ap₂A or Ap₃A, evokes tear secretion (Pintor et al., 2002a,b), suggesting that these two dinucleotides play a role in the regulation of corneal hydration and cleaning.

The mechanisms that control and regulate intraocular pressure are not fully understood, but it results from the dynamic equilibrium between the production and drainage (or resorption) of aqueous humor. In general terms, sympathetic activity results in a reduction in intraocular pressure, and parasympathetic activity results in an increase. It seems that the main point of control of intraocular pressure is the outflow of aqueous humor, rather than its production (Burke and Potter, 1986; Judge and Flitcroft, 2000; Jumblatt, 2000). Intraocular pressure may also be regulated by effectors of circadian rhythms, and we have recently shown that melatonin and the MT₃ receptor ligand, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT or GR 135531), can cause profound decreases in pressure (Pintor et al., 2001).

In the present experimental work, we describe the presence of diadenosine polyphosphates in the aqueous humor and the differential effects of a series of diadenosine polyphosphates, Ap₂A, Ap₃A, Ap₄A, and Ap₅A, which includes those known to be present in tears, on intraocular pressure in the rabbit. Our results suggest that one of them, Ap₄A, has the potential to be used a therapeutic agent in conditions where intraocular pressure is elevated.

	Materials and Methods

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Animals. New Zealand White rabbits (males, 2-3 kg) were used. The animals were kept in individual cages with free access to food and water, under controlled 12-h light/dark cycles. Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research.

Aqueous Humor Collection and Sample Preparation. New Zealand White rabbits were anesthetized with 1.5 mg·kg¹ propofol (Abbott Laboratories, Madrid, Spain). Aqueous humor was removed with a syringe connected to a 30-gauge needle in the sclero-corneal limbus area. Samples were stored at 35°C before treatment. The treatment consisted of their chromatography through a SEP-PAK Accell QMA cartridges (Waters, Milford, MA) and elution of the retained nucleotides and dinucleotides by means of a mixture of 0.1 N HCL 0.2 N KCl. Eluates were neutralized with 10 N KOH before HPLC injection (Rotllán et al., 1991).

HPLC Procedures. The HPLC system consisted of a Waters 1515 isocratic HPLC pump, a 2487 dual absorbance detector and a Reodyne injector, all managed by the software Breeze from Waters. The column was a Novapak C-18 (15 cm length, 0.4 cm diameter) from Waters.

Intraocular Pressure Measurements. Intraocular pressure was measured by means of a Tono-Pen XL contact tonometer (Mentor Massachusetts Inc., Norwell, MA). This device has been shown to be the tonometer of choice for measuring intraocular pressures within the range of 3 to 30 mm Hg in rabbits (Abrams et al., 1996). All measurements fell within this diapason: the mean baseline value of intraocular pressure was 17.0 ± 0.39 mm Hg (n = 112). A topical anesthetic [Colircusí Laboratorios Cusí, Madrid, Spain (0.1 mg · ml¹ tetracaine plus 0.4 mg · ml¹ oxybuprocaine in 0.9% saline, diluted 1:3 in 0.9% saline)] was applied (10 µl) to the cornea before each measurement of intraocular pressure was made (Pintor et al., 2001). Diadenosine polyphosphates or saline was applied topically to the cornea in volumes of 10 µl.

Drugs Used. Diadenosine pyrophosphate sodium salt, diadenosine triphosphate ammonium salt, diadenosine tetraphosphate ammonium salt, and diadenosine pentaphosphate sodium salt, ,-meATP, suramin, and RB-2 were all obtained from Sigma-Aldrich. ATPS was purchased from Roche Diagnostics (Mannheim, Germany). Pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, was obtained from Sigma/RBI (Natick, MA). All the dinucleotides were dissolved in sterile saline (0.9%), to produce stocks with concentrations in the range from 1 ng · ml¹ to 1 mg · ml¹. Suramin, RB-2, and PPADS were diluted to produce a stock of 1 mg · ml¹.

Analysis of Data. Numerical values are given as mean ± S.E.M. Means of two groups were compared using Student's t test (unpaired, two-tailed, unless stated) with a 5% fiducial point of significance. Means of three or more groups were compared using analysis of variance and post hoc Tukey's tests, using  = 0.05.

	Results

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Presence of Diadenosine Polyphosphates in the Aqueous Humor. The analysis of nucleotide and dinucleotide content in rabbit aqueous humor indicated the presence of several peaks, two of which were tentatively identified as Ap₄A and Ap₅A, when compared to commercial standards (Fig. 1). To confirm the nature of the putative dinucleotides, samples were enriched with commercial dinucleotides that coeluted together with the peaks present in the samples (results not shown). To fully confirm the existence of Ap₄A and Ap₅A in the aqueous humor, peaks were collected and submitted to phosphodiesterase treatment. This enzyme cleaves the dinucleotide giving AMP plus another nucleotide that depends on the length of the phosphate chain in the original dinucleotide. When diadenosine tetraphosphate was submitted to this treatment, it was possible to observe the presence of AMP and ATP (Fig. 2, upper traces); Ap₅A digestion with phosphodiesterase gave AMP and adenosine 5'-tetraphosphate (Ap₄) as products (Fig. 2, lower traces). This treatment therefore confirmed that the two dinucleotide peaks present in the aqueous humor corresponded to Ap₄A and Ap₅A. The concentrations of these dinucleotides were then calculated by comparison with samples of commercial external standards. Ap₄A and Ap₅A were present in the aqueous humor at concentrations of 0.34 ± 0.1 and 0.08 ± 0.01 µM, respectively (n = 8).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Presence of diadenosine polyphosphates in rabbit aqueous humor. Samples analyzed as described under Materials and Methods presented two putative peaks (upper trace) identified tentatively as Ap4A and Ap5A when compared with comercial standards (lower trace, 1000 pmol each). Insert, blow-up of the putative adenine dinucleotide peaks.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Phosphodiesterase treatment and analysis of putative diadenosine polyphosphates. Diadenosine polyphosphates were treated with phosphodiesterase from C. durissus as described under Materials and Methods. The treatment of the putative Ap4A (upper trace) completely changed the chromatographic profile with peaks corresponding to AMP and ATP appearing as digestion products (second trace). When the same treatment was carried out on the putative Ap5A, two peaks, AMP and Ap4 appeared as digestion products (lower traces).

Effect of Diadenosine Polyphosphates on Intraocular Pressure. Ap₂A and Ap₃A both caused a dose-dependent increase in intraocular pressure (Figs. 3 and 4). The threshold dose was between 0.1 and 1.0 µg · 10 µl¹. The dose-response relationships did not appear to saturate, there being no peak or plateau in the dose-response curves before the highest dose (300 µg · 10 µl¹). Ap₅A also caused increases in intraocular pressure. However, a dose-response relationship was not clear. At doses below 10 µg · 10 µl¹ responses were extremely variable, with a coefficient of variability greater than the value of the mean (data not shown). At doses of 10, 100, and 300 µg · 10 µl¹, increases in pressure were clear, but lacked dose dependence (Fig. 4)

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of instillation of Ap2A into the rabbit eye on increased IOP. Points show mean ± S.E. of eight tests.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of instillation of Ap3A and Ap5A into the rabbit eye on IOP at doses of 10, 100, and 300 µg · 10 µl1. Bars show mean ± S.E. of eight tests.

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View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of instillation of Ap4A into the rabbit eye on IOP. Points show mean ± S.E. of eight tests.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Time course of changes in IOP in response to instillation of adenine dinucleotides. A, Ap2A (closed square, n = 8); Ap3A (open square, n = 8) at 100 µg · 10 µl1; saline (10 µl, vehicle, open triangle, n = 8). B, Ap4A (open circle, n = 8); Ap5A (closed circle, n = 8), all at 100 µg · 10 µl1; saline (10 µl, vehicle applied to contralateral eye, open triangle, n = 16). *, P < 0.05, **, P < 0.01 relative to t30 min.

Cross-Desensitization and Antagonist Studies. To study the receptors that mediate the actions of the diadenosine polyphosphates, cross-desensitization experiments with nucleotides were performed.

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View larger version (24K): [in this window] [in a new window]   Fig. 7.   Cross-desensitization experiments with different nucleotides. A, homologous and heterologous desensitization experiments for Ap4A (at 100 µg · 10 µl1). ,-meATP and ATPS were preincubated as described under Materials and Methods at concentrations of 100 µg · 10 µl1. B, cross-desensitization experiments for Ap5A in the presence of ,-meATP and ATPS (all at 100 µg · 10 µl1). In all the experiments n = 8.

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View larger version (25K): [in this window] [in a new window]   Fig. 8.   Experiments with P2 receptor antagonists. A, antagonism of suramin, RB-2, and PPADS (all at 100 µg · 10 µl1) on the hypotensive response induced by Ap4A (at 100 µg · 10 µl1). B, antagonism on the hypertensive effect induced by Ap5A by suramin, RB-2, and PPADS (all at 100 µg · 10 µl1). In all the experiments n = 8. *, P < 0.05, relative to dinucleotide responses.

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Antagonism of responses to Ap2A, Ap3A, Ap4A, and Ap5A by PPADS (100 µg · 10 µl1). For each pair of columns, the left-hand column shows the effect of the corresponding dinucleotide on IOP in the absence of PPADS, while the right-hand column shows the effect of the dinucleotide in the presence of PPADS. Bars show mean ± S.E. of eight experiments. *, P < 0.05 relative to t30 min.

	Discussion

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These results show, for the first time, the presence of diadenosine polyphosphates in the aqueous humor of that New Zealand White rabbits. Chromatographic analysis of these dinucleotides demonstrated that Ap₄A and Ap₅A are present in the aqueous humor in the low micromolar range. Adenine mononucleotides were also identified in the aqueous humor, and they may also participate in the regulation of the IOP as previously indicated (Pintor and Peral, 2001).

Our results also show that topical application of diadenosine polyphosphates to the cornea can have profound effects on intraocular pressure in the rabbit. Ap₂A, Ap₃A, and Ap₅A all produced increases in pressure, while Ap₄A produced a decrease. Cross-desensitization studies and antagonism with suramin, PPADS, and RB-2 indicate that the polyphosphates were acting via P2 receptors, although the subtypes involved have not been fully characterized.

Ap₄A was a potent agonist and produced a decrease in intraocular pressure at concentrations 3 orders of magnitude below those at which Ap₂A, Ap₃A, or Ap₅A produced an increase. The dose-response curve for Ap₄A did not appear to inflect at the highest concentrations tested points at which activation of the excitatory receptor might be expected. At the lowest concentrations tested, none of Ap₂A, Ap₃A, and Ap₅A produced a decrease in intraocular pressure, which implies that in addition to there being two separate populations of receptors, one mediating an increase and the other a decrease in intraocular pressure, this latter receptor is specific for Ap₄A. It is possible that Ap₄A also activates the excitatory receptor, but in the mixed population the effects of activation of the receptor that mediates a decrease in pressure predominated. In this sense, cross-desensitization studies suggest that Ap₄A acts through the same receptor as ,-meATP (a P2X receptor), while Ap₅A acts to the same as ATPS (a P2Y receptor) (Pintor and Peral, 2001). It has not been possible to identify the molecular mechanisms that link these two receptors and the physiological processes that control IOP.

Ap₄A and Ap₅A are also present in rabbit tears (Pintor et al., 2002b), and both stimulate tear secretion in rabbits, as does Ap₆A, while Ap₂A and Ap₃A do not (Pintor et al., 2002b). Their presence in tears and aqueous humor and their effects on both tear secretion and intraocular pressure suggests that they are serving physiological roles in maintaining corneal hydration and eye pressure. The endogenous tear concentration of Ap₄A is close to 3 µM (Pintor et al., 2002b), and this is within its range of activity for reducing intraocular pressure when topically applied as a single 10-µl dose. The IC₅₀ concentration was equivalent to 13 µM. It is important to point out that the concentrations of diadenosine polyphosphates determined in the aqueous humor may reflect a more physiological concentration than those that occur when the dinucleotides are applied topically. Diadenosine polyphosphates may be partially hydrolyzed when passing through the cornea before reaching the aqueous humor, therefore the real IC₅₀ values for these compounds in the eye anterior chamber should be lower than that apparently obtained.

It is unusual to find a tissue or organ in which members of the homologous series of diadenosine polyphosphates, with a chain length from two to five, have opposing actions. However, there are examples of receptors that are selective or specific for one or members of this group of compounds. For example, receptors that are activated by Ap₄A but not other diadenosine nucleosides have been described in recombinant rat P2X₂ receptors, expressed in Xenopus laevis oocytes. This receptor is activated by Ap₄A, but not Ap₂A, Ap₃A, or Ap₅A (Pintor et al., 1996; Wildman et al., 1999). The MM39 cell line, derived from human tracheal gland epithelium, possesses a native receptor that is also sensitive to Ap₄A but not Ap₃A or Ap₅A (Saleh et al., 1999). The P2 receptor on rat mast cells (the P2Z receptor, which opens a membrane pore) is activated by Ap₄A, but not other dinucleotides. This is because Ap₄A can bear four negative charges, one per phosphate group, whereas smaller dinucleotides cannot, and larger dinucleotides do not because they chelate divalent cations (Tatham et al., 1988). In contrast receptors that are activated by Ap₂A, Ap₃A, and Ap₅A but not Ap₄A have not been identified with any clarity.

P2X receptors in the guinea pig vas deferens and urinary bladder are insensitive to Ap₂A, and Ap₃A is only a weak agonist (Hoyle et al., 1995). Similarly at P2X receptors in the rat mesenteric arterial bed Ap₄A, Ap₅A, and Ap₆A are all agonists, while Ap₂A and Ap₃A are not (Ralevic et al., 1995). In contrast, Ap₂A and Ap₃A are both good agonists of P2Y receptors in the guinea pig taenia coli and rat mesenteric arterial endothelial cells (Hoyle et al., 1995; Ralevic et al., 1995; Hourani et al., 1998), and in ECV304 cells, a cell line derived from human umbilical endothelial cells, there is a P2Y receptor that is sensitive to Ap₃A but not Ap₄A, Ap₅A, or Ap₆A (Conant et al., 1998).

In conclusion, diadenosine polyphosphates are present in the rabbit aqueous humor, moreover, intraocular pressure can be regulated by topical corneal application of adenine dinucleotides. Ap₄A potently decreases intraocular pressure; Ap₂A, Ap₃A, and Ap₅A increase intraocular pressure. The receptors involved remain to be characterized, but the one activated by Ap₄A bears similarities to some previously described P2X receptors. The fact that these adenine dinucleotides modulate intraocular pressure leads to the suggestion that they may be useful compounds in the development of therapeutic agents. In particular, Ap₄A, or a derivative, may be useful in the treatment of ocular disorders such as forms of glaucoma, in which a reduction of intraocular pressure would be beneficial.